The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Determination of the genomic sequence of higher organisms, including humans, is now an attainable goal. However, this analysis represents only one aspect of the information encoded by the genome. Genes are expressed in an ordered and timely manner, and also exhibit a precise spatial and temporal expression pattern. Consequently, knowing the sequence of the genome is insufficient to explain biology and to understand disease.
More significantly, genes are transcribed to messenger RNA, which is then translated to protein. It is the protein, or gene product, that exhibits activity, and carries out the work of the cell. With the post-genome era rapidly approaching, new strategies for the analysis of proteins are being developed. Most conventional approaches focus on recording variations in protein level. These approaches are commonly referred to as “proteomics”. In general, proteomics seeks to measure the abundance of broad profiles of proteins from complex biological mixtures.
In the most common embodiments, proteomics involves separating the proteins within a sample by two-dimensional electrophoresis. Then, the individual protein spot patterns of these gels can be compared to get indications as to the relative abundance of a particular protein in two comparative samples. The approach can even be extended to determine the molecular identity of the individual protein spots by excising the spots and subjecting them to peptide mass fingerprinting. For example, U.S. Pat. No. 6,064,754, which is hereby incorporated by reference in its entirety, including all figures, tables, and claims, describes computer-assisted methods for separating biomolecules and identifying biomolecule subsets by comparing profiles of the biomolecules obtained in gel electrophoretic separations.
More recently, methods have been described for eliminating the electrophoresis steps and performing proteomics by directly analyzing the complex mixture by mass spectrometry. For example, methods currently described in the art provide chemically reactive probes that can be reacted with a protein mixture to label many proteins in that mixture in a non-specific, or non-directed, manner providing a quantitative analysis only of protein abundance (see Aebersold, PCT/US99/19415). Such methods disclose that there are many chemically reactive amino acid residues within a protein which are individually reactive and which can be conjugated to chemical probes, whereby protein conjugates can be subsequently quantified to yield an indication of protein abundance Similarly, Wells et al. (PCT/US99/14267; PCT/US98/21759) disclose methods for identifying small organic molecule ligands that bind to biological target molecules without the requirement that the ligand bind to an active site on a target molecule. See also, WO 00/00823 and WO 00/11208.
Workers have also described methods for profiling classes of proteins based on protein activity using “activity-based probes” or “ABPs.” In these methods, molecules with a binding moiety directed to the active site of a given protein class (e.g., serine proteases) and linked to a biotin tag are used to differentiate active members of the protein class in a proteome from inactive members. See, e.g., Liu et al., Proc. Nat'l. Acad. Sci. USA 96: 14694–14699 (1999); Cravatt and Sorensen, Curr. Opin. Chem. Biol. 4: 663–668 (2000); Patricelli et al., Proteomics 1: 1067–71 (2001). Each of these references is hereby incorporated in its entirety.
With regard to analysis or proteins using fluorescent labels, Scholze et al., Anal. Biochem. 276: 72–80 discloses fluorescent inhibitors for analysis of lipases; U.S. Pat. No. 4,433,051 discloses an enzyme-activated irreversable inhibitor of omithine decarboxylase linked to a rhodamine moiety for use in cytochemical staining procedures. U.S. Pat. No. 6,127,134, which is hereby incorporated by reference in its entirety, including all figures, tables, and claims, discloses the analysis of protein mixtures using fluorescent compounds and separation using electrophoresis.
In determining active proteins in a complex protein mixture, the goal is typically to compare different assay compositions, so that one can relate the different compositions for better understanding of the nature of the protein mixture. There remains a need in the art for methods and compositions that permit different compositions to be accurately compared as to the presence and/or abundance of each of the different active proteins in the mixture.